Abbreviations to be used in this description are as follows.
2DSH: 2-O-desulfated HEP
2SH: (6-O.N)-desulfated-N-acetylated HEP
6DSH: 6-O-desulfated HEP
6SH: (2-O.N)-desulfated-N-acetylated HEP
Ac-2DSH: N-acetylated 2DSH
Ac-6DSH: N-acetylated 6DSH
Ac-NAH: N-acetylated NAH
Ac-NSH: N-acetylated NSH
CDSH: Completely desulfated-N-acetylated HEP
Ch: Chondroitin
CS-A(S): Chondroitin sulfate A derived from shark
CS-A(W): Chondroitin sulfate A derived from whale
CS-A: Chondroitin sulfate A
CS-B: Chondroitin sulfate B
CS-C: Chondroitin sulfate C
CS-D: Chondroitin sulfate D
CS-E: Chondroitin sulfate E
EHS-HS: Heparan sulfate derived from Engelbreth-Holm-Swarm tumor tissue
GAG: Glycosaminoglycan
HA: Hyaluronic acid
HEP: Heparin
HS: Heparan sulfate
HSPG: Proteoglycan-heparan sulfate
IdoA: L-iduronic acid
KS: keratan sulfate
NAc-HEP: N-acetylated HEP
NAH: N-acetyl heparosan
NDST: Glucosaminyl N-deacetylase/N-sulfotransferase
NH2-2SH: (6-O.N)-desulfated HEP
NH2-6SH: (2-O.N)-desulfated HEP
NH2—CDSH: Completely desulfated HEP
NH2—HEP: N-desulfated HEP
NSH: (2-O.6-O)-desulfated HEP
In this description, D-glucosamine residue is represented as GlcNH2; hexuronic acid residue is represented as HexA; N-sulfated-D-glucosamine residue is represented as GlcNS; D-glucuronic acid residue is represented as GlcA; N-acetyl-D-glucosamine residue is represented as GlcNAc; and partially de-acetylated NAH is represented as PDNAc-NAH.
HS and HEP, which are kinds of GAGs, are acidic polysaccharides having a disaccharide-repeated structure containing a D-glucosamine residue and a hexuronic acid (GlcA or IdoA) residue as a basic sugar chain structure. In addition, they are modified by O-sulfation, N-sulfation, or N-acetylation to various degrees. HS is present as HSPG in cell surfaces of almost all animals (Non-patent Document 1), and is a major component of an extracellular matrix.
On the other hand, HEP is localized in a mucosal and a granule in a mast cell. As results of in vitro experiments, it has been revealed that HEP binds to many bioactive proteins (hereinafter, referred to as “HEP-binding proteins” or “HBPs”) and that the functions of such HBPs are regulated by binding HEP. For example, HEP is considered to stabilize HBPs such as fibroblast growth factors, hepatocyte growth factor, etc., and to act as a cofactor or a co-receptor, thereby controlling cell proliferation. Further, the same results were confirmed in the experiments using HS. The structure of HEP that binds to each bioactive HBP is not uniform, and the importance of the degree of sulfation or site-specific sulfation is being recognized.
A major ligand of HBPs in vivo is considered to be not HEP but HS. That is, HS is considered to regulate the above-mentioned various biological reactions. Therefore, HS that is widely present in animal bodies and has various functions is important component for animals and human. From biological and medical points of view, it is very important to acquire qualitative and quantitative method for precisely detecting HS. Accordingly, a monoclonal antibody against HS may be a unique detection reagent.
Because of the aforementioned reasons, many reports on production of antibodies using HS or HEP as an antigen have been made in recent years. mAb HepSS-1 and mAb F58-10E4, which are monoclonal antibodies that recognize an N-sulfated glucosamine unit of HS (-[HexA-GlcNS]-), are commercially available and widely used.
When the structure of HS is analyzed in accordance with the method of “2.8 Structural analysis using degrading enzyme of glycosaminoglycan and HPLC in combination” described in Non-patent Document 2, it is easily understood that a major component disaccharide of HS is a nonsulfated disaccharide, GlcA-GlcNAc. The ratio reaches 50 to 60% although it depends on an organ from which HS is isolated. That is, the major structure of HS is an N-acetylglucosamine unit (-[GlcA-GlcNAc]-).
Meanwhile, in a certain disease such as diabetes, NDST activity that catalyzes a first step of the sulfation reaction of HS and HEP is decreased, resulting in increase in the ratio of an N-acetylglucosamine unit (Non-patent Document 3). Further, HS derived from kidney is known to contain a glucosamine unit (-[GlcA-GlcNH2]-) (Non-patent Document 4). Therefore, in order to distinguish HS derived from kidney from HSs derived from other organs, it is desirable to use an antibody that specifically recognizes a structure present in HS derived from kidney, that is, a glucosamine unit. Accordingly, detection/quantification of the N-acetylglucosamine unit or glucosamine unit, which forms a nonsulfated domain of HS, is as important as detection/quantification of a sulfated domain.
However, monoclonal antibodies that recognize the glucosamine unit or N-acetylglucosamine unit, which form a nonsulfated domain of HS, are only mAb JM403 and mAb 865 (Non-patent Documents 4 to 7), respectively.
mAb JM-403, which recognizes a glucosamine unit of HS, has been prepared from a mouse that was immunized with HSPG purified from rat glomerulus by van den Born, J. et al. van den Born, J. et al. have reported that HS contains 2 to 3 glucosamine units in average, and that the reactivity of the antibody disappears by acetylation of HS. They have also reported that the antibody does not react with NAH but react with de-N-acetylated NAH more strongly as compared to HS. In addition, they have reported that an N-sulfated glucosamine unit present in HS does not affect the reactivity of the antibody, but an O-sulfated structure or an IdoA structure inhibits the reactivity of the antibody (Non-patent Document 8).
On the other hand, mAb 865, which recognizes an N-acetylglucosamine unit of HS, was originally obtained as an antibody against enterobacterial common antigen (anti-ECA) by Peters, H. et al., and is cross-reactive to a capsular polysaccharide of Escherichia coli K5 (hereinafter, referred to as “NAH”). van den Born, J. et al. has reported that the antibody does not react with sulfated NAH or N-deacetylated NAH and its epitope is an N-acetylglucosamine unit of 18 sugars or more. Meanwhile, the reactivity of the antibody to HS is 1/3,000 or less than that to NAH (Non-patent Document 7), and it has been reported that the antibody reacts with mouse EHS-HS, which has high sulfation degree as compared to normal HS.
Mouse EHS-HS is HS with high sulfation degree. Therefore, an antibody capable of reacting with mouse EHS-HS reacts with HS of high sulfation degree, so the antibody is considered to have relatively low specificity to the antigen. As described above, an antibody that recognizes nonsulfated region containing N-acetylglucosamine units, glucosamine units, and the like of HS but does not react with mouse EHS-HS was not known, and therefore such an antibody has been desired.
Non-patent Document 1: Biochemistry, 10, 20 1445 (1971)
Non-patent Document 2: Shin Seikagaku Jikken Koza 3, Carbohydrates II (published by Tokyo Kagaku Dojin Co., Ltd., 1991) p. 49-62
Non-patent Document 3: Diabetes, 40, 1449 (1991)
Non-patent Document 4: Kidney Int., 41, 115 (1992)
Non-patent Document 5: J. Biol. Chem., 270, 31303 (1995)
Non-patent Document 6: Infect. Immun., 50, 459 (1985)
Non-patent Document 7: J. Biol. Chem., 271, 22802 (1996)
Non-patent Document 8: J. Biol. Chem., 270, 31303 (1995)